Cell line for the expression of an α2δ2 calcium channel subunit and methods of use

ABSTRACT

Described is a method for determining the binding ability of a compound to bind to an α2δ2 subunit of a calcium channel comprising: providing an α2δ2 subunit of a calcium channel, contacting the subunit with the compound, and determining the binding ability of the compound to bind to the subunit.

This application claims the benefit of PCT/US01/14799 filed May 8, 2001,which claims the benefit of U.S. Provisional Application 60/204,466filed May 16, 2000; the entire contents of each of which are herebyincorporated herein by reference.

FIELD OF THE INVENTION

This invention pertains to cell lines which express an α2δ2 subunit of avoltage-dependent calcium channel, where the cell lines may also expressadditional calcium channel subunits, and where the binding ofgabapentin, gabapentin analogues, pregabalin, or pregabalin to the cellsmay be determined.

BACKGROUND OF THE INVENTION

Voltage-dependent calcium channels have been linked to physiologicalprocesses such as neurotransmitter release, secretion of hormones,muscle contraction, and regulation of gene transcription. A functionalchannel requires at least three subunits, including the α1, α2δand βsubunits. The channel may also contain a γ subunit. There are severalknown types of voltage-dependent calcium channels that have been definedbased on their electrophysiological characteristics and pharmacologicalproperties. These types are L-, N-, P/Q-, R-, and T-type. Each type isprimarily defined by its channel composition. The type of α1 subunitcontained in the channel determines whether the channel is an L-, N-,P/Q-, R-, or T-type channel. The activity of the α1 subunit is modulatedby the α2δ and β subunits. Channel activity may be further modulated bya fourth subunit, γ.

Molecular biological techniques have allowed elucidation of themechanism of voltage-dependent calcium channel action. Genes for each ofthe subunits have been isolated and cloned. There are currently nineknown genes encoding for different α1 subunits. The a1 subunit forms thepore which calcium ions flow through. The α1 subunit contains thevoltage sensor and is also responsible for the binding specificity ofcertain drugs or toxin that may be associated with the channel type.Channel current through the α1 pore may be modulated by association ofthe β, γ, or α2δ subunit. There are four known genes for theintracellular β subunit that may be differentially spliced. There aretwo known genes for the transmembraneγ subunit, one in skeletal muscleand a novel gene expressed in the brain. Only one isoform of α2δ wasinitially identified. Recently, however, two new α2δ genes wereidentified, α2δ2 and α2δ3. These genes have 55.6 and 30.3% homology withthe original α2δ1 gene (Klugbauer, et al., J Neuroscience1999;19(2):684–691). The α2 and δ proteins are expressed by the samegene. The protein product is post-translationally cleaved, and thefinal. α2 and δ proteins are linked by disulfide bonds. Thetransmembrane δ protein secures the α2 protein to the cell membrane.

Studies have shown that the α2δ1 subunit contains a binding site for theanticonvulsant drug, gabapentin [1-(aminomethyl)cyclohexane acetic acid](Gee, et al., J. Biol. Chem. 1996;271(10):5768–5776). Gabapentin is aγ-aminobutyric acid (GABA) analogue. Gabapentin is effective in thetreatment of epilepsy and in decreasing seizure frequency in both animalmodels and in human patients. The precise mechanism of action ofgabapentin remains unclear. Recent experiments have shown thatgabapentin also binds to the α2δ2 subunit.

Functional channels may be formed by expression of the calcium channelsubunits in a cell. This technique is advantageous in determining theeffects of various molecules on channel action. U.S. Pat. No. 5,712,158and U.S. Pat. No. 5,770,447 describe a stable cell line that is usefulfor investigating gabapentin binding properties to calcium channelsubunits. This cell line expresses the β subunit and the original α2δsubunit (now referred to as α2δ1) at high levels. Transfecting the cellswith any α1 subunit results in the formation of functional calciumchannels which can be used to evaluate the binding of gabapentin andgabapentin-related compounds.

It is the object of this invention to provide a new cell line thatstably expresses a calcium channel α2δ2 subunit. It is a further objectof this invention to describe α2δ2 subtype-specific binding ofgabapentin, analogues of gabapentin, pregabalin, analogues ofpregabalin, and 3-alkyl derivatives of GABA.

SUMMARY OF THE INVENTION

The invention provides a method for determining the binding ability of acompound to an α2δ2 subunit of a calcium channel comprising: providingan α2δ2 subunit of a calcium channel, contacting the α2δ2 subunit withthe compound, and determining the binding ability of the compound to theα2δ2 subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrams the molecular cloning of human α2δ2 into the pCDNA3.1expression vector.

FIG. 2. RT-PCR Analysis of Human α2δ Tissue Distribution. One ng ofdouble-stranded cDNA from different human tissues (CLONTECH) wasamplified by PCR with 35 cycles of 94° C. for 1 minute, 55° C. for 1minute, and 72° C. for 2 minutes. The generated PCR products representDNA fragments from nucleotide 958 to 2165 (hα261), 2534 to 3643 (hα2δ2),and 1920 to 3272 (hα2δ3).

FIG. 3A–F Northern Blot Analysis Human α2δ Tissue Distribution. Northernblotting was carried out as described in Materials and Methods. Humanmultiple tissue blots (CLONTECH) were hybridized withDigoxigenin-labeled cDNA synthesized from nucleotide 958 to 2165 (hα2δ1), 2534 to 3643 (h α2δ2), and 1920 to 3272 (h α2δ3). The positionsof marker RNA are indicated to the left.

FIG. 4A–B Western Blot Analysis of Human and Mouse .alpha.2.delta.Tissue Distribution. Membrane proteins from different human tissues (A,0.5 μg) and mouse tissues (B, 100 μg) were loaded on 4% to 20% SDS-PAGE(NOVEX) and subjected to Western blot analysis (see Materials andMethods). The blots were probed with anti-α2δ monoclonal antibody orpolyclonal antibodies against α2δ2 and α2δ3.

FIG. 5. Binding of [³H] Gabapentin to Membranes From COS-7 CellsTransfected With α2δ cDNA. COS-7 cells were transfected with 20 μg ofpcDNA3.1 (control), pcDNA3.1/porcine α2δ1 construct (pα2δ1), andpcDNA3.1 //human α2δ2 construct (hα2δ2). The membranes were prepared for[3H]gabapentin binding assays (see Materials and Methods). Data are anaverage of three independent assays with triplet in each determination.The same membranes (100 μg) were subjected to Western blot analysis withcorresponding antibodies as described in Materials and Methods.

FIG. 6. Disruption of Disulphide-Linkage Between α2 and δ Subunits. Anequal amount of membrane protein from each sample (0.5 μg for pα2δ1 and5 μg for hα2δ2) was incubated in the presence or absence of 100 mM DTTfor 10 minutes and resolved on a nonreducing SDS-PAGE and transferred toa PVDF membrane. The blots were probed with either an anti-α261 antibody(left) or an anti-α2δ2 antibody (right). The positions of markerproteins are indicated to the right.

FIG. 7. Scatchard Analysis of [³H]gabapentin (GBP) Binding to MembranesForm HEK293 Cells Overproducing Porcine α2δ1 (A) and Human α2δ2 (B). Thecell membranes were prepared from GKS02, a stable cell line for porcineα2δ1, and GKS07, a stable cell line for human α2δ2. The specific[³H]gabapentin binding was carried out as described in Materials andMethods. The binding activity was expressed as pmole of gabapentin boundper mg of protein. Each binding reaction contained 20 μg of GKS02membrane proteins or 10 μg of GKS07 membrane proteins. Data wereaverages of three assays.

FIG. 8. Screening Cell Lines by [³H] Gabapentin (GBP) Binding Activity.HEK293 cells were transferred with human α2δ2. Single clones wereselected by G418-resistance. “2923,” parental cells HEK293; “2L,” BEK293cells stably expressing porcine α2δ1.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, analogues of gabapentin include but are not limited toalkyl-substituted gabapentin analogues, bridged gabapentin analogues,and heterocyclic gabapentin analogues such as those described by Bryans,et al. in J. Med. Chem. 1998;41:1838–1845. Analogues are defined as“compounds with similar electronic structures but different atoms”(Grant, et al., Chemical Dictionary, 5th ed., McGraw-Hill, 1987).Gabapentin has the structure:

Examples of gabapentin analogues are described in Bryans, et al., supra,and include, but are not limited to:

A molecule with the structure:

This analogue is alkylated at position 3 on the cyclohexane ring. Ananalogue may be alkylated at any position on a carbon ring with an alkylgroup of from 1 to 4 carbon atoms. An analogue may also be a moleculewith the structure:

This analogue is alkyl-substituted at the 3-position of the gabapentinring. Molecules of this type include pregabalin

its analogues, and 3-alkyl derivatives of GABA.

MATERIALS AND METHODS

Porcine α2δ1 (pα2δ1) cDNA was from J. Brown (Brown J. P., Dissanayke V.U. K., Briggs A. R., Milic M. R, Gee N., Anal. Biochem.,1998;255:236–243). Mouse α2δ3 (mα2δ3) cDNA was a generous gift from F.Hoffman (Klugbauer N., Lacinova L., Marais E., Hobom M., Hofmann F., J.Neuroscl, 1999;19:684–691). Monoclonal antibody against α2δ1 waspurchased from Affinity Bioreagents, Inc. Polyclonal antibodies againstα2δ2 and α2δ3 were from Sandra Duffy (Pfizer). Human and mouse multipletissue blots and cDNA were purchased from CLONTECH. Mouse tissues werepurchased from Pet-Freez Biologicals. PCR reagents were from Invitrogen.ECL Western blot kit was from Armersham. Lipofectamine, growth media,restriction enzymes were from LifeTechnologies. HEK293 and COS-7 celllines were from ATCC. All other chemicals were from Sigma.

Cloning of human α2δ2 subunit. Human α2δ2 (hα2δ2) cDNA was amplified byPCR from a human brain cDNA library. Based on the deposited DNA sequenceof hα2δ2 subunit from GenBank (accession number AF042792), fouroverlapped DNA fragments, which covered the whole open reading frame ofhα2δ2 cDNA from nt −14 to 994 (fragment H), 845 to 1816 (fragment F),1517 to 2791 (fragment D), and 2681 to 3790 (fragment C), were generatedby PCR and then cloned into expression vector pcDNA3.1 by TA cloningkit. The sequences of the primer pairs used were:

5′-TCTTGAATGGAAACATGGCGGTGC-3′ SEQ ID No. 1) and

5′-TATACCAGGGTCTCCTTCGGACAT-3′ SEQ ID No. 2) (fragment H);

5′-ATGTGTTCATGGAAAACCGCAGAC-3′ SEQ ID No. 3) and

5′-AGCCGTTCAGGTCAATGGCAAACA-3′ SEQ ID No. 4) (fragment F);

5′-CCATCCGCATCAACACACAGGAAT-3′ SEQ ID No. 5) and

5′-GTAAGTCCTCATTGTTAACCTCGC-3′ SEQ ID No. 6) (fragment D);

5′-CTGAGAAGTTCAAGGTGCTAGCCA-3′ SEQ ID No. 7) and

5′-GATGTGATTTGGGTGCCAAACACC-3′ SEQ ID No. 8) (fragment C). The fourfragments were cut at internal unique restriction enzyme sites at nt 791(PflM I), 1395 (Xba I), and 2628 (Hpa I), and assembled into pcDNA3.1vector (Invitrogen, Carlsbad, Calif.) at Hind III/Xho I sites (see FIG.1).

RT-PCR. Double-stranded cDNA preparations from different tissues(CLONTECH) were used for PCR reaction with 35 cycles at 94° C. for 1minute, 55° C. for 1 minute, and 72° C. for 2 minutes. The reactionswere performed in a solution containing 1 ng cDNA, 10 pM primers, 1 mMdNTPs, and 1×PCR buffer in a volume of 50 μL. Ten microliters of thereaction mix was loaded on 1% agarose gel. The primer pairs for humanα8δ1, α2δ2, and α6δ3 were

5′-GACGCGGTGAATAATATCACAGCC-3′ SEQ ID No. 9) and

5′-ACAAATCGTGCTTTCACTCCCTTG-3′ nt 958 to 2165; accession number M76559)(SEQ ID No. 10);

5′-CTGAGAAGTTCAAGGTGCTAGCCA-3′ SEQ ID No. 11) and

5′-GATGTGATTTGGGTGCCAAACACC-3′ nt 2534 to 3643; accession numberAF042792) (SEQ ID No. 12); and

5′-CGTGTCCTTGGCAGATGAATGGTC-3′ SEQ ID No. 13) and

5′-CATCTCAGTCAGTGTCACCTTGAG-3′ nt 1920 to 3272; accession numberAJ272213) (SEQ ID No. 14), respectively. The expected lengths of PCRproducts from human α8δ1, α2δ2, and α6δ3 were 1208, 1110, and 1352 bp.These primers were specific for each subtype of α2δ as determined bysequencing analysis of the corresponding PCR products.

Northern blot analysis. Multiple Tissue Northern Blots (CLONTECH) werehybridized and washed according to the manufacturer's recommendation.Digoxigenin-labeled probes specific for subtypes of α2δ were generatedby PCR and hybridized in 10 mL EasyHyb (Boehringer Mennhaim) at 50° C.overnight. The same pairs of primers as those used for RT-PCR wereemployed to generate the probes. The blots were washed twice, first in2×SSC and 0.1% SDS at room temperature for 5 minutes, then in 0.1×SSCand 0.1% SDS at 68° C. for 15 minutes. Detection of expression was inaccordance with the manufacturer's instructions (Boehringer Mennhaim).

Cell culture and transfection. COS-7 and HEK293 cells were cultured inDMEM and RPMI 1640 media, respectively. The media were supplemented with50 units/mL penicillin, 50 μg/mL streptomycin, and 10% heat-inactivatedfetal bovine serum (FBS), in a humidified incubator with 95% air and 5%CO₂ at 37° C. For transient transfection into COS-7 cells, 20 μg ofplasmid DNA (vector or the same vector with α2δ insert) was incubatedwith 30 μL of lipofectamine. The mixture was overlaid onto the cells in1.5 mL serum-free medium and incubated for 5 hours. Then FBS was addedto the dishes to bring the final concentration to 10%. The medium waschanged next morning. Forty-eight hours after the transfection, thecells were harvested for membrane preparation. For stable transfectionof porcine α2δ1 and human α2δ2 into HEK 293 cells, the same procedurewas applied as that for a transient transfection except for that 800μg/mL G418 (gentacin) was added to the cells 48 hours after thetransfection. Two clones, GKS02 and GKS07, showed highest expression ofporcine α2δ1 and human α2δ2, respectively, and were selected for furtherbinding studies. The cell line has ATCC No. PTA-1823. In addition, hostsfor expression of α2δ2 protein binding assays can also includeeukaryotic expression systems such as yeast, insect cells, and mammaliancells (CHO, COS-7, HEK293, etc.).

Membrane preparation. Membranes were prepared from tissues or culturedcells. The cells were washed twice with cold PBS and then scraped offwith cold buffer containing Tris (5 mM, pH 7.4), EDTA (5 mM), PMSF (0.1mM), leupeptin (0.02 mM), and pepstatin (0.02 mM). The cells wereincubated on ice for 30 minutes, followed by sonication for 30 to 40seconds. For membrane preparations from tissues, the tissues were slicedinto small pieces and subjected to sonication at interval of 10 seconds4 times. The resulting homogenates from tissues or cultured cells werecentrifuged for 10 minutes at 750 to 1000×g, and then the supernatantswere centrifuged at 50,000×g for 30 minutes. The resulting pellets wereresuspended in the same buffer as described above.

Western blot analysis. The cell membranes (0.5 μg for GKS07 cells, 5 μgfor GKS02 cells, 100 μg for transiently transferred cells or tissues)were resolved by 4% to 20% SDS-PAGE and transferred to nitrocellulosemembranes using semi-dry transferring unit. The membranes were incubatedwith either rabbit anti-α2δ1, α2δ2, and α2δ3 antibodies for 1 hour atroom temperature, followed by washing with 1×PBS. The blots wereincubated with anti-rabbit IgG for 1 hour and developed with ECLreaction according to the procedure recommended by manufacturer.

Binding assays. The radioligand-binding assay was done using membraneproteins incubated in the presence of 20 nM [³H]gabapentin. Themembranes (100 μg of proteins for transiently transfected cells, 20 μgfor GKS02 cell membranes, and 10 μg for GKS07 cell membranes) wereincubated in 10 mM HEPES(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) (pH 7.4) for40 to 50 minutes at room temperature, and then filtered onto pre-wettedGF/C membranes and quickly washed five times with 3 mL of ice cold 50 mMTris buffer (pH 7.4). The filters were dried and counted in a liquidscintillation counter. For determining nonspecific binding, the bindingassays were performed in the presence of 10 μM pregabalin (Gee N. S.,Brown J. P., Dissanayake V. U., Offord J.; Thurlow R., Woodruff G. N.,J. Biol. Chem., 1996;271:5768–5776). The specific binding was obtainedby subtracting nonspecific binding from the total binding. Clone #7 wasidentified as the highest α2δ2 subunit expressing clone. Binding assayscan also be performed using recombinant and/or purified α2δ2 proteinfrom human and other mammalian species, for screening α2δ2subtype-selective inhibitors.

RESULTS

Tissue distribution of α2δ transcripts. Tissue distribution of hα2δ1,hα2δ2, and hα2δ3 mRNA was first examined by RT-PCR analysis. Theseprobes were designed to specifically amplify three subtypes of α2δ. Asshown in FIG. 2, single PCR products corresponding well to the predictedsizes of hα2δ1, hα2δ2, and hα2δ3 (1208, 1110, and 1352 bp) appeared inalmost all tissues tested. A much higher level of hα2δ2 transcript wasfound in lung than any other tissues including brain. Since the PCRproducts showed sequences identical to the corresponding α2δ, the widescope of tissue distribution revealed the ubiquitous feature of hα2δmRNA expression. However, the RT-PCR condition used here did not yieldquantitative estimation of 2δ mRNA levels among different tissues,Northern analysis is necessary for estimating the relative abundance ofeach subtype of hα2δ mRNA. Northern blots demonstrated that all threehα2δ genes were expressed about equally well in brain, heart, andskeletal muscle except for the much higher expression of hα2δ1 inskeletal muscle (FIG. 3). In addition to these three tissues, the mostabundant hα2δ2 transcript was found in lung. The highest expression ofhα2δ2 mRNA in lung was consistent with the above described RT-PCRresults and also agreed well with one recent report (Gao B., Sekido Y.,Maximov A., Saad M., Forgacs E., Latif F., et al., J. Biol. Chem.,2000;275:12237–12242), but differed from an early observation (KlugbauerN., Lacinova L., Marais E., Hobom M., Hofmann, F., J.Neurosci.,1999;19:684–691). In the present study we also detected asmall amount of hα2δ1 and hα2δ3 mRNAs in liver and kidney, respectively.Results from this and other laboratories (Klugbauer, Supra., 1999; Gao,Supra., 2000, and our unpublished data) have shown that expression ofmouse α2δ3 (mα2δ3) is restricted to the brain. The expression of hα2δ3also in tissues other than brain suggested species difference in 2δ3expression.

In the brain, hα2δ1, hα2δ2, and hα2δ3 were detected in every portions ofbrain tissues tested including cerebellum, cerebral cortex, medulla,occipital pole, frontal lobe, temporal lobe, and putamen. A higher levelof hα2δ2 transcript was found in cerebellum than cerebral cortex, whilereverse was true for hα2δ3. For hα2δ1, its mRNA was approximatelyequally distributed in these two regions. The expression patterns of thethree isoforms in these two brain regions were in accordance withprevious in situ hybridization results (Klugbauer, Supra., 1999; HobomM., Dai S., Marais E., Lacinova L., Hofmann F., Klugbauer N., Eur. J.Neurosci., 2000;12:1217–1226). In addition, all three subtypes of 2δmRNA were found in spinal cord, but at lower levels than that found inthe brain.

Tissue distribution of 2δ proteins. Although the level of protein isfunction of the steady-state level of mRNA, the relative abundance ofmRNA and protein of specific gene is not always proportional, which mayreflect post transcriptional regulation (Jackson V. N., Price N. T.,Carpenter L., Halestrap A. P., Biochem. J., 1997;324:447–453). Toexamine the relative levels of human and mouse 2δ subunits acrosstissues, we used antibodies raised against specific subtypes of 2δprotein for Western analysis. Equal amounts of proteins were loaded onSDS polyacrylamide gels. Consistent with the ubiquitous distribution ofhα2δ1, Western blots of human and mouse tissues showed that both hα2δ1and mα2δ1 proteins were widely distributed, although hα2δ1 in lung andjejunum were not detectable. By contrast, hα2δ3 protein was onlydetected in brain, not in lung, testis, aorta, spleen, jejunum, andkidney (FIG. 4A). Similarly, mα2δ3 protein was found only in brain, notin heart, kidney, liver, lung, pancreas, stomach, spleen thymus, ovary,pituitary, thyroid, and prostate. Surprisingly, in contrast topredominant expression of hα2δ2 transcript in lung (FIGS. 2 and 3),hα2δ2 protein was predominantly found in brain and the level of hα2δ2protein was not detectable in lung (FIG. 4A). In addition to brain, lowlevels of hα2δ2 protein were also found in aorta, testis, andventricular muscle. There seemed to be two immunoreactive bands intestis with one equivalent to predicted molecular weight of hα2δ2 (175kDa) and the other showing slightly lower molecular weight. This lowermolecular protein appeared to be similar to the predominant banddetected in ventricular muscle. As previously observed with pα2δ1, thislower band may represent the dissociated α2 subunit from the α2δ proteinor an isoform of α2δ2 (Brown J. P., Dissanayke V. U. K., Briggs A. R.,Milic M. R., Gee N., Anal. Biochem., 1998;255:236–243; Wang M., OffordJ., Oxender D. L., Su, T. Z., Biochem. J., 1999;342:313–320). Inaddition, two immunoreactive bands were also detected in mouse heart byanti-α2δ2 antibodies, but the predominant band in this case hadmolecular weight higher than that found in other tissues (FIG. 4B).

Disulphide linkage of α2 and δ proteins. It has been shown that α2 and δsubunits of α2δ1 were linked by disulphide bond (Wang, Supra., 1999).Since the amino acid sequence in δ region is less conserved between α2δ1and α2δ2, it is interesting to know if α2δ2 protein is also cleaved intotwo subunits post translation. To examine such a possibility, cellmembranes from HEK 293 cell lines overproducing pα2δ1 (GKS02) and hα2δ2(GKS07) proteins were treated or untreated with 100 mM DTT before gelelectrophoresis. In the presence of DTT, both pα2δ1 and hα2δ2 proteinswere shifted to a position predicted for α2, suggesting that as withpα2δ1, hα2δ2 also consists of two subunits that are linked by disulphidebond (FIG. 6).

[³H]Gabapentin Binding. To determine the gabapentin binding propertiesof the cloned hα2δ2, membranes were isolated from COS-7 cellstransiently transfected with pα2δ1, hα2δ2, and vector pcDNA3.1.Expression of the corresponding α2δ proteins was examined by Westernblots. As shown in FIG. 5, transfection of the cells with pα2δ1 resultedin a prominent increase in gabapentin binding. Similarly, the cellsexpressing hα2δ2 exhibited about fourfold increase in gabapentin-bindingactivity. Although a slightly increased binding; activity was observedin the cells transfected with pcDNA3.1 vector alone, statistic analysisdid not show that this smaller change was significant.

Gabapentin bindingK_(D) and the binding properties of pα2δ1 and hα2δ2were determined in cell lines GSK02 (pα2δ1) and GKS07 (hα2δ2). In HEK293cells stably expressing pα2δ1, [³H]gabapentin bound to a singlepopulation of sites as demonstrated in previous report (Gee, Supra.,1996) withK_(D) value of 72±9 nM (FIG. 7A). Similarly, a singlepopulation of binding sites were also observed in hα2δ2-containingmembranes (FIG. 7B), but theK_(D) value was higher than that of pα2δ1(156±25 nM). To determine pharmacological properties of hα2δ2, severalcompounds were selected for competition with [³H]gabapentin binding. Asimilar, but not identical profile of competition was seen in the twosubtypes of α2δ protein (Table 1). For instance, binding to bothsubtypes of α2δ were stereo-selective because L-leucine was markedlymore potent than its D-enantiomer. The affinities of BCH, a modelsubstrate of system L transport (Su T. Z., Lunney E., Campbell G.,Oxender D. L., J Neurochem., 1995;64:2125–2131), and phenylalanine wereweak for both subtype proteins. On the other hand, gabapentin binding toα2δ2 was more sensitive to (S+)-3-isobutyl GABA (pregabalin) with IC₅₀value of 96 nM as compared to 149 nM for pα2δ1.

TABLE 1 IC₅₀ Values for Inhibition of [³H]Gabapentin Binding toMembranes From Stable Cell Lines Overproducing Porcine α2δ1 (GKS02) andHuman α2δ2 (GKS07) by Selected Amino Acids Compounds GKS02 (pα2δ1) GKS07(hα2δ2) Gabapentin 132 282 Pregabalin 149 96 L-leucine 118 205L-phenylalanine 825 2,960 D-leucine 198,960 151,510 BCH 1,028 775

FIG. 8 also illustrates the screening of stable cell lines that expresshuman α2δ2 protein.

While the forms of the invention herein disclosed constitute presentlypreferred embodiments, many others are possible. It is not intendedherein to mention all of the possible equivalent forms or ramificationsof the invention. It is understood that the terms used herein are merelydescriptive, rather than limiting, and that various changes may be madewithout departing from the spirit or scope of the invention.

1. A method for determining the binding ability of a compound to a cellexpressing an α2δ2 subunit of a calcium channel comprising providing acell line expressing an α2δ2 sub-unit of a calcium channel, said cellline having ATCC No. PTA-1823; contacting the cells with the compound;and determining the binding ability of the compound to the cells.
 2. Themethod of claim 1 wherein the compound is gabapentin.
 3. The method ofclaim 1 wherein the compound is a 3-alkyl substituted gabapentin.
 4. Themethod of claim 1 wherein the compound is pregabalin.
 5. The method ofclaim 1 wherein the compound is a 3-alkyl derivative of y-aminobutiricacid (GABA).
 6. A cell line having ATCC No. PTA-1823.
 7. A method fordetermining the binding ability of a compound to an α2δ2 subunit of acalcium channel comprising; providing an α2δ2 subunit of a calciumchannel encoded by the nucleotide sequence set forth in accession NO. AF042792; contacting the α2δ2 subunit with the compound; and determiningthe binding ability of the compound to the α2δ2 subunit.
 8. The methodof claim 7 wherein the compound is gabapentin.
 9. The method of claim 7wherein the compound is a 3-alkyl substituted gabapentin.
 10. The methodof claim 7 wherein the compound is pregabalin.
 11. The method of claim 7wherein the compound is a 3-alkyl derivative of GABA.
 12. The method ofclaim 7 wherein the α2δ2 subunit is a purified protein.